Extensive genetic studies have established that the fidelities of replicative DNA polymerases and their associated proofreading 3'-exonucleases are the primary determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have developed a bacteriophage system that permits the rapid analysis of the fidelities of mutant polymerases in vivo without a requirement for enzyme over-expression, purification, and fidelity analysis in vitro (although the latter option remains available). Bacteriophage RB69 DNA polymerase (whose structure has been described) supplied from a plasmid can replace the normal bacteriophage T4 DNA polymerase when the latter is mutationally inactivated. In this system, high fidelity is retained during T4 DNA replication. Using mutations that alter critical polymerase and exonuclease amino acids, we are characterizing the resulting changes in mutation rates using both reversion and forward-mutation tests in vivo and forward-mutation tests in vitro. Either inactivating the proofreading exonuclease or altering key residues in or near the polymerase active site strongly increases mutation rates.